Since the introduction of various vaccines against SARS-CoV-2 at the end of 2020, infection rates have continued to climb worldwide. This led to the establishment of a third dose vaccination in several countries, known as a booster. To date, there has been little real-world data about the immunological effect of this strategy.We compared the humoral- and cellular immune response before and after the third dose of BioNTech/Pfizer vaccine BNT162b2, following different prime-boost regimen in a prospective observational study. Humoral immunity was assessed by determining anti-SARS-CoV-2 binding antibodies using a standardized quantitative assay. In addition, neutralizing antibodies were measured using a commercial surrogate ELISA-assay. Interferon-gamma release was measured after stimulating blood-cells with SARS-CoV-2 specific peptides using a commercial assay to evaluate the cellular immune response.We included 243 health-care workers who provided blood samples and questionnaires pre- and post- third vaccination. The median antibody level increased significantly after the third vaccination dose to 2663.1 BAU/ml vs. 101.4 BAU/ml (p < 0.001) before administration of the booster dose. This was also detected for neutralizing antibodies with a binding inhibition of 99.68% ± 0.36% vs. 69.06% ± 19.88% after the second dose (p < 0.001). 96.3% of the participants showed a detectable T-cell-response after the booster dose with a mean interferon-gamma level of 2207.07 mIU/ml ± 1905 mIU/ml.This study detected a BMI-dependent antibody increase after the third dose of BNT162b2 following different vaccination protocols. All participants showed a significant increase in their immune response. This, in combination with the low rate of post-vaccination-symptoms underlines the potential beneficial effect of a BNT162b2-booster dose. Following an initially flattened curve of COVID-19 infections, due to vaccination campaigns all over the world, there came a subsequent resurgence of COVID-19 infections worldwide , 3 and dAs the impact of different vaccines on the immune system varies, considering both cellular and humoral immunity is crucial. The detected levels of neutralizing antibodies have been shown to be higher after administration of the BioNTech/Pfizer vaccine BNT162b2 than those after receiving the AstraZeneca vaccine ChAdOx1, although this could not be shown for T-cell responses . These dWe aimed to evaluate the humoral- and cellular immune response after a third booster-dose of BNT162b2. The booster dose was given following an initial administration of either two doses of BNT162b2 (Group 1), two doses of ChAdOx1 (Group 2) or cross-vaccination of BNT162b2+ChAdOx1 (Group 3) in a real-world setting analyzing one of the most important groups in this global pandemic – health care workers.th – 14th 2021 .To evaluate the efficiency of the third dose, all participants were asked to provide an additional blood sample and complete a questionnaire 4 weeks after the booster-vaccination  to stay in accordance with the WHO standard. Antibodies were determined by using a fully automated quantitative anti-SARS-CoV-2-assay (IgG) from Abbott . This is a chemiluminescent microparticle immunoassay (CMIA) which measures the IgG antibodies against the spike receptor-binding domain (RBD) of SARS-CoV-2. As recommended by the manufacturer, a value below 7.1 BAU/ml was determined to be negative whereas values ≥ 7.1 BAU/ml were determined to be positive.To evaluate the neutralizing anti-SARS-CoV-2 antibodies, all samples were additionally tested using the NeutraLISA™ SARS-CoV-2 Neutralization Antibody Detection KIT . Results were presented in binding inhibition, where values ≥ 35% were considered positive. In contrast to the widely established neutralization assays using cell-cultures, this ELISA-based surrogate assay can be easily be performed in all laboratories and has showed a good correlation to the known cell-culture based assays . Even a In addition to the humoral immune response, the cellular immunity to SARS-CoV-2 was also assessed using the Quan-T-cell SARS-CoV-2 kit  working as an Interferon-gamma release assay (IGRA). The analysis for this Quan-T-cell test was performed as previously reported . IFN-gamIn the initial phase of this study in April 2020, participants completed a questionnaire regarding their medical history, including current medication and smoking habits. Before and after administration of the third dose of vaccine, all participants were asked to complete a questionnaire regarding their prior vaccinations, current smoking behaviors, and in addition after the third vaccination dose, possible side effects related to the booster dose. Specifically, participants were questioned about localized symptoms  and systemic symptoms, such as fever or fatigue.This study was conducted in accordance with the Declaration of Helsinki. All participants provided written and informed consent before inclusion. This study was prospectively registered at the German Clinical Trial Register (DRKS00021270) after approval by the Ethics Committee of the Medical Association Schleswig-Holstein.All variables are presented as means or medians with standard deviation or interquartile range. Categorical variables are shown as numbers with percentages. Statistical analysis was made using IBM SPSS Statistics Version 25 . In addition, GraphPad Prism 9 was used for graphics.Relationships between categorial variables were tested using the Chi-square test or the Fisher’s exact test, depending on the size of the groups. Inter-group differences were analyzed using Mann-Whitney-U test or Kruskal-Wallis-test reporting the mean with standard deviation. Pre- and post-third dose antibody and INF-gamma levels were compared using Wilcoxon sign rank test. A p-value < 0.05 was considered statistically significant.To investigate the joint effect of age, sex, body mass index (BMI) and current smoking on the relative increase of antibody-level, a linear regression analysis was performed to take the antibody level before booster into account. The IgG-levels had a skewed distribution and was logarithmized for the regression analysis yielding an approximately normal distribution. As the dependent variable, the difference of the logarithmized IgG-levels after and before booster was used. The backward selection method was used to determine the final model.Before administration of the third vaccine-dose, 310 participants provided a blood specimen, of which 243 resubmitted a blood sample and completed questionnaire exactly 4 weeks after the booster dose (follow-up-rate 78.39%).The participants included 59 (24.3%) male and 184 (75.7%) female participants with a mean age of 46.33 ± 11.44 years. The characteristics of the study cohort are described in All participants received a booster dose of BNT162b2, irrespective of their primary vaccination protocol. The participants included 179 (73.7%) individuals who had previously received two doses of BNT162b2 (Group 1), 50 (20.6%) individuals who had received heterologous vaccinations with BNT162b2+ChAdOx1 (Group 2), 12 (4.9%) individuals who were double vaccinated with ChAdOx1 (Group 3), and 2 (0.8%) individuals who had a natural SARS-CoV-2 infection followed by a single dose of BNT162b2.Prior to the booster dose, all participants still showed an antibody level above the manufacturer’s cutoff (> 7.1 BAU/ml). The median antibody level increased significantly after the third dose, when the whole study cohort was considered . All individuals showed increased antibody-levels with a median increase of 2539.55, IQR 1613.2-4002.5   (Anti-SARS-CoV-2 binding antibody levels differed significantly between the subgroups (representing different prior vaccination protocols), prior to booster-administration. In brief, pre-boost antibody levels were highest in Group 2 (183.1 BAU/ml), followed by Group 1 (124.9 BAU/ml) and Group 3 (52.75 BAU/ml). The administration of a third BNT162b2 dose led to a highly significantly increased antibody-level, across all analyzed subgroups. Inductive effects were highest within Group 3 participants , p = 0.002), followed by Group 1 , p < 0.001) and Group 2 , p < 0.001) participants Table 2.Neutralizing antibody levels also differed significantly between the groups prior to booster-administration Table 2.Prior to the administration of a booster BNT162b2 dosage, SARS-CoV-2 specific IFN-gamma release of stimulated blood-cells significantly differed between individuals of each considered group Table 2.2 value of 0.43. The regression coefficients remained virtually unchanged when only the significant variables, BMI and antibody-level before booster, remained in the final model  had a prior infection, no conclusion can be drawn from this result. al model Table 3.We compared the immune status before the third dose showing that obese participants have a lower mean anti-SARS-CoV-2 binding antibody level in comparison with the non-obese participants . This was also seen for the neutralizing antibodies .From the regression model, the estimated antibody-level showed both an absolute and relative increase given the BMI and the antibody-level before booster. The majority of the study cohort reported some minor side-effect following the booster dose Table 1.We present real-life data showing the impact of administration of a third dose of COVID-19 vaccination using BNT162b2 mRNA vaccine, following different vaccination protocols in a well-defined group of health care employees. Our data reveal a significantly increased cellular and humoral immune response to this additional vaccination dose.Our data show that both humoral- and cellular immunity appear to be most persistent after double ChAdOx1 vaccination compared to the other vaccination strategies included in the study. In contrast, Dulovic et al. in their study showed that the persistence of neutralizing antibodies is shortest in individuals who have received the homologous ChAdOx1 vaccine. Contrarily, the administration of a heterologous- or a homologous mRNA strategy led to a stronger persistence. These discrepancies can be explained by the fact that Dulovic et al. only examined persistence over a maximum period of 65 days following administration of the second vaccine dose . In our Our study shows the highest increase in INF-gamma release, binding-antibody expression, as well as neutralizing antibody capability for participants initially vaccinated two times with ChAdOx1. However, these participants showed the weakest humoral immune response before administration of the booster dose. A regression analysis detected a high BMI to be associated with an increased immune response.Several studies have analyzed the effect of a third dose on the immune response and on the occurrence of infections, and showed an overall beneficial effect of the third dose, without relevant adverse events , 24–29.Atmar et al. analyzed in their phase 1-2 open-label clinical trial the efficiency of homologous and heterologous protocols for the third dose, and found a stronger increase in immunity against SARS-CoV-2 following a heterologous third dose . This isIn addition to the determined overall binding antibody titers, the neutralizing antibodies play a main role in the humoral immune response and are correlated with protection against severe COVID-19 outcome .Using the commercial ELISA-based NeutraLISA assay from Euroimmun, our data show a substantial increase in neutralizing antibody capability in all participants after booster-administration. This is in line with the data presented from Atmar et al. . Data frMultiple studies have tried, and are still attempting, to evaluate possible factors associated with a reduced immune response or with a complete non-response to anti-SARS-CoV-2-vaccination.Within the study cohort presented here, smoking, increased age and BMI have previously been shown to have a negative impact in studies involving two doses of BNT162b2  or 9 monOther factors potentially influencing the immune response after a third dose of BNT162b2 are immunosuppression, for instance in patients after solid-organ transplantation .In our cohort, we could not detect any negative impact of older age, although elderly participants are relatively underrepresented in this trial. Data published by Eliakim-Raz et al. also described no correlation to higher age in a group older than 60 years .These findings can aid in the development of a safe vaccination protocol, especially for patients with a potentially higher risk, for example because of a high BMI. It also provides real world data following different vaccination protocols, and helps to develop benchmarks for a potential immune response.Further studies are required to provide more accurate information on risk factors associated with reduced immunity to SARS-CoV-2.Despite the fact this study benefits from its real-life data in an important group of health-care workers, and a relatively large number of participants, it comes with some limitations.This study is limited by its single-center design. The inclusion of health-care workers led to the overrepresentation of women and younger people, whereas groups with a higher risk for low immune response to vaccination, such as elderly and participants with an immunomodulatory treatment, are underrepresented. To ensure a good comparability of the samples, we performed the follow-up within two days. This led to a lost-to-follow-up proportion of 21.6% of the previously screened participants, due to holidays or different working schedules. Unfortunately, the number of individuals in each group of vaccination protocols were unequally distributed, as there was no individual choice regarding the vaccine received. This led to a very small number of participants in Group 3, limiting the reported strong immune response. As this was designed as a serological study, the survey of side-effects was not in the main focus and is limited due to the retrospective evaluation. Additionally, this study is limited by the use of an ELISA-based surrogate neutralization test. Even this showed a good correlation to the gold standard of a cell-culture based neutralization assay, the testing against new variants is not possible. As this study aims to report the short-term-outcomes of the third dose within the COVID-19-vaccination protocol, the long-term effect, especially for debatable factors as the BMI, are missing.This study showed a BMI-dependent increase in antibody titers after the third dose of BNT162b2 following different vaccination protocols, although all participants were noted to have a significant increase in their immune response. In this study, participants after two doses of ChAdOx1 who received BNT162b2 as a third dose showed the strongest immune response. This effect, in combination with mild-to-none post-vaccination symptoms, underlines the potential beneficial effect of a BNT162b2-booster dose following 2 doses of anti-SARS-CoV-2-vaccine. Further studies are needed to evaluate thresholds for the immune response – both humoral and cellular – and to detect the longevity of the booster-induced immunity.The raw data supporting the conclusions of this article will be made available by the authors on reasonable request, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Medical Association Schleswig-Holstein, Germany. The participants provided their written informed consent to participate in this study.JH and BF conducted the research and wrote the manuscript. These authors contributed equally to this research. HB supervised the statistical analysis. A-KB, HH, and SYG reviewed the manuscript. TS initiated the research and supervised the study. TS and CK reviewed the final draft of this article and provided logistic support. All authors have critically reviewed and approved the final draft and are responsible for the content of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In the 7th post-injury month, rats underwent continuous vEEG monitoring to detect spontaneous seizures and assess sleep disturbances. At the end of the experiments, rats were perfused for brain histology. Results: In the TBI group, the percentage of rats with PTZ-induced seizures increased over time (adjusted p < 0.05 compared with D30). Combinations of three PTZ test parameters (latency to the first epileptiform discharge (ED), number of EDs, and number of PTZ-induced seizures) survived the leave-one-out validation for differentiating rats with or without epileptiform activity, indicating an area under the receiver operating curve (AUC) of 0.743  with a misclassification rate of 36% on D90, and an AUC of 0.752  with a misclassification rate of 32% on D180. Sleep analysis revealed that the number of transitions to N3 or rapid eye movement (REM) sleep, along with the total number of transitions, was increased in the TBI group during the lights-on period . The sleep fragmentation index during the lights-on period was greater in the TBI rats than in sham-operated rats (p < 0.05). A combination of sleep parameters showed promise as diagnostic biomarkers of prior TBI, with an AUC of 0.792  and a misclassification rate of 28%. Rats with epilepsy or any epileptiform activity had more transitions from N3 to the awake stage (p < 0.05), and the number of N3–awake transitions differentiated rats with or without epileptiform activity, with an AUC of 0.857 . Combining sleep parameters with PTZ parameters did not improve the biomarker performance. Significance: This is the first attempt to monitor the evolution of seizure susceptibility over months in a well-described rat model of PTE. Our data suggest that assessment of seizure susceptibility and sleep disturbance can provide diagnostic biomarkers of prior TBI and prognostic biomarkers of post-traumatic epileptogenesis.Objectives: We investigated whether seizure susceptibility increases over weeks–months after experimental traumatic brain injury (TBI), and whether seizure susceptibility in rats predicts the development of post-traumatic epilepsy (PTE) or epileptiform activity. We further investigated whether rats develop chronic sleep disturbance after TBI, and whether sleep disturbance parameters—alone or in combination with pentylenetetrazol (PTZ) test parameters—could serve as novel biomarkers for the development of post-traumatic epileptogenesis. Methods: TBI was induced in adult male Sprague-Dawley rats with lateral fluid-percussion injury. Sham-operated experimental controls underwent craniectomy without exposure to an impact force. Seizure susceptibility was tested with a PTZ test  on day (D) 30, D60, D90, and D180 after TBI ( Approximately 70 million individuals are estimated to suffer a TBI each year . TBIs acEpileptogenesis refers to the development and extension of tissue capable of generating spontaneous seizures, resulting in (a) the development of an epileptic condition and/or (b) progression of epilepsy after it is established . After sTo date, the results of cross-sectional studies have identified promising blood, imaging, and electroencephalogram (EEG) biomarkers of epileptogenesis after TBI ,12. HoweAdministration of a subconvulsive dose of the chemoconvulsant pentylenetetrazol (PTZ) is widely used to evaluate seizure susceptibility in genetically modified mice, inbred mice and rats with spontaneous seizures, and animals exposed to various epileptogenic brain injuries, including TBIs, as well as to identify antiseizure medications . The PTZHere, we tested the hypothesis that PTZ-induced seizure susceptibility increases over a period of weeks to months after experimental TBI. We further evaluated whether a clear increase in seizure susceptibility observed in rats could predict the development of PTE or epileptiform activity. In addition, as late unprovoked seizures and epileptiform activity typically occur at the transition between N3 and rapid eye movement (REM) sleep , we inveThe study design is summarized in n = 50, Harlan, The Netherlands) were used (weight 321 ± 27 g on the day of surgery). Rats were housed in individual cages and maintained in a controlled environment . Water and food were available ad libitum. Experiments were approved by the Committee for the Welfare of Laboratory Animals of the University of Eastern Finland. All of the experiments were conducted according to the European Community Council Directives 2010/63/EU.Adult male Sprague-Dawley rats (n = 16) or TBI (n = 34) groups, as summarized in A total of 50 rats were randomized to the sham operation (On day (D) 21 after TBI or sham operation (D0 was marked as the day of injury), rats were anesthetized as described above and inserted into the stereotaxic apparatus. Two cortical stainless steel screw electrodes  were inserted over the parietal cortex, targeting the S1 area —one rostral to the craniotomy, and the other at the corresponding coordinates contralaterally A,B. Two Long-term vEEG monitoring was performed to (a) assess the induced epileptiform activity in the PTZ seizure-susceptibility tests on D30, D60, D90, and D180, (b) detect unprovoked seizures, i.e., diagnose PTE during the 7th post-injury month, (c) record other types of epileptiform activity during the 7th post-injury month, and (d) monitor sleep during the 7th post-injury month.Details of the methodology were described previously in . BrieflyDeficits in the somatomotor and vestibular functions were assessed by the composite neuroscore test, as previously described in . TestingTo monitor the evolution of post-TBI seizure susceptibility, rats underwent a PTZ seizure-susceptibility test on D30, D60, D90, and D180 after TBI. Rats were connected to the vEEG system 24 h before the PTZ test, and monitoring was continued for 24 h after the test, after which the rats were disconnected from the monitoring system . A subcoA seizure was defined as a high-amplitude rhythmic discharge with frequency and amplitude modulation that clearly represented an abnormal EEG pattern  and lasted at least 10 s. An electrographic ED was defined as a rhythmic high-amplitude discharge, containing a burst of slow waves, spike-and-wave, and/or poly-spike-and-wave components, and lasting < 10 s. Spikes were defined as high-amplitude electrographic elements with a duration between 20 and 70 ms.On D180, rats were reconnected to the vEEG for 30 days to monitor the occurrence of unprovoked epileptiform activity . SeizureA 24-h epoch on the 15th day of a 30-day recording during the 7th post-TBI month was analyzed. In case of the occurrence of seizures or a poor-quality EEG recording, a 24-h EEG epoch starting 48 h earlier was included in the analysis. Sleep was scored as recommended by the American Academy of Sleep Medicine, with slight modifications . BrieflyTo assess the location and extent of the FPI and exclude non-TBI-related epileptogenic lesions , rats were perfused for histology after completing the vEEG (D210).Perfusion: Briefly, rats were deeply anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg), and perfused transcardially via the aorta with 0.9% NaCl , followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 . The brain was removed from the skull, fixed in 4% paraformaldehyde for 4 h, cryoprotected in 20% glycerol in 0.02 M potassium phosphate-buffered saline (pH 7.4) for 24 h, frozen in dry ice, and stored at −70 °C for further processing.Frozen coronal sections of the brain were cut  using a sliding microtome. The first series of sections was stored in 10% formalin at room temperature and used for thionin staining. Other series of sections were collected into tissue collection solution  and stored at −20 °C until processed.®  as a mounting medium.Nissl staining: The first series of sections was stained with thionin, cleared in xylene, and coverslipped using DePeXhttps://unfoldedmap.org, accessed on 16 April 2022) that was adapted to the rat brain [Preparation of unfolded cortical maps: To assess the cortical lesion area and damage to different cytoarchitectonic cortical areas after TBI, all thionin-stained sections were digitized . Unfolded cortical maps were then prepared from the digitized histologic sections as previously described in , by applat brain .https://github.com/brian-lau/MatlabAUC, accessed on 16 April 2022). The 95% confidence interval for the cross-validated AUC was estimated using a bias-corrected and accelerated bootstrap method. In all tests of statistical significance, a p value < 0.05 was considered significant. Data are shown as the mean ± standard deviation of the mean (unless otherwise specified).Statistical analyses were performed using SPSS for Windows (v. 27.0). Treatment and time effects on body weight, composite neuroscore, and the PTZ seizure-susceptibility test were analyzed using repeated-measures analysis of variance (ANOVA), followed by post hoc Bonferroni correction for multiple tests. Within each treatment group, the evolution of changes was assessed using Friedman’s 2-way ANOVA or a related-samples Cochran’s Q test, followed by post hoc Bonferroni correction for multiple tests. Intra-timepoint comparisons between the animal groups were tested with the nonparametric Kruskal–Wallis test, followed by post hoc analysis with the Mann–Whitney U test . Cohen’s delta was assessed as an indicator of effect size. The sensitivity and specificity of the single PTZ test and sleep parameters were assessed by receiver operating characteristic (ROC) analysis. To assess the presence of model overfitting, the logistic regression fitting and ROC analysis were repeated using a leave-one-out cross-validation, implemented in MATLAB . The cross-validated area under the ROC curve (AUC) was estimated using the pooling method  and BriaAcute (<48 h) post-impact mortality was 15% (5/34) in the TBI group and 0% (0/16) in the sham-operated group. Follow-up mortality due to unknown causes was 3% (1/29). Thus, 16 sham-operated and 28 TBI rats underwent the PTZ seizure-susceptibility test, followed by 1 month of continuous vEEG monitoring .p > 0.05) and 89% in the TBI group . Over the follow-up, the body weight remained slightly lower in the TBI group than in the sham group (p < 0.001). In both groups, the body weight increased after the first week compared with that on D2 (Changes in body weight over the 180-day follow-up are summarized in at on D2 A.p < 0.001). Although rats with TBI showed somatomotor recovery from D2 to D7 (p < 0.001), they did not reach the performance levels of sham-operated experimental controls during the 3-week post-injury follow-up.The severity of the post-TBI somatomotor impairment and recovery over the 21-day follow-up is summarized in Monitoring: Forty-four rats  completed the PTZ test follow-up and 1-month video EEG monitoring . Of the Occurrence of epileptiform activity: Altogether, 15 of the remaining 22 TBI rats showed epileptiform activity in EEG (TBIe+ group) and 7 did not (TBIe−) , includiWe also observed some epileptiform activity in 3 of the 16 sham-operated animals during the 7th month after the sham operation (craniectomy), including spikes within N3 spindles, periodic EDs, and trains of large-amplitude sharp waves.Occurrence of unprovoked seizures: Four of the twenty-two TBI rats had at least one unprovoked electrographic seizure over the 4-week monitoring during the 7th month post-TBI (TBI+), and eighteen did not (TBI−). That is, 18% (4/22) of the monitored TBI rats had developed PTE A. Rat #4EEG quality was sufficient for analysis of induced and unprovoked epileptiform activity in all 16 sham cases and 28 TBI cases. The PTZ-induced seizure-susceptibility test data are summarized in The Mann–Whitney U test revealed no differences between the TBI and sham groups in any of the PTZ test parameters on D30, D60, D90, or D180 . HoweverSham-operated experimental controls: We observed no increase in seizure susceptibility over time in the sham group .p < 0.001, related-samples Cochran’s Q test)     A. The nurection) . The latrection) . The latrection) .p < 0.05) and on D180   .n = 7), the latency to the first ED decreased from 976 ± 833 s on D30 to 207 ± 71 s on D90  (In the TBIe− group (rection) .n = 7), latency to the PTZ-induced first seizure decreased from 370 ± 209 s on D30 to 144 ± 87 s on D90  (In the TBIe+ group (rection) .p = 0.061, Friedman’s 2-way ANOVA with Bonferroni correction; The TBI+ group contained only four animals (see below). No differences were detected between the TBI+ and TBI−groups at any of the testing points . Howeverp > 0.05). Data are summarized in A total of 5 animals in the sham group and 14 in the TBI group were excluded from the sleep analysis due to noisy EEG channels. Thus, sleep EEG was analyzed in 11 sham-operated and 14 TBI rats . The avep > 0.05). As expected, the total duration of sleep was shorter during the lights-off period than during the lights-on period (12 h/12 h) in both the sham group  and the TBI group . The duration of sleep was comparable between the sham and TBI groups during the lights-off period (sham 5.58 h ± 0.89 h vs. TBI 5.14 ± 0.57 h) and the lights-on period (sham 9.04 ± 0.71 h vs. TBI 9.00 ± 0.78 h)  for approximately 60% of the 24-h recording time . No diff 0.78 h) .p < 0.01). Moreover, when the lights-on and lights-off periods were combined, the average duration of REM periods was shorter in the TBI group than in the sham-operated rats .Duration of different sleep–awake stages: Next, we assessed the duration of different sleep–awake stages during the lights-off and lights-on periods . During p < 0.05). Moreover, the number of transitions to the REM stage was greater in the TBI group than in the sham group . Overall, the total number of transitions during the lights-on period was greater in the TBI group than in the sham group , and when the lights-on and lights-off periods were combined   .Total duration of sleep: The duration of awake and sleep periods during the lights-on or lights-off periods did not differ between the TBIe+ and TBIe−groups .p < 0.05), as well as when the lights-on and lights-off periods were combined  (p < 0.05). The duration of REM sleep was shorter in the TBIe+ group than in the TBIe− group during the lights-on period .Duration of different sleep–awake stages: The average duration of N2 periods was shorter in the TBIe+ group than in the TBIe− group during the lights-off period  . The avep < 0.05), during the lights-off period , and when the lights-on and lights-off periods were combined   .Total duration of sleep: The duration of the awake and sleep periods during the lights-on and lights-off periods did not differ between the TBI+ and TBI− groups .p < 0.05)  .p < 0.05), as well as when the lights-on and lights-off periods were combined   .In the same 24-h epoch that was used for the sleep disturbance analysis, we also analyzed the occurrence of spike-and-wave discharges (SWDs), which are described as normal oscillations in many rat strains .p > 0.05) in EEGs recorded during the 7th post-injury month. On average, the number of SWDs per 24-h EEG epoch was 127 ± 96  in the sham rats and 127 ± 133  in the TBI group (p > 0.05). In both groups, SWDs occurred exclusively during the awake stage  and 9 (64%), respectively, showed SWDs  and 192 ± 145 in rats with epileptiform activity .TBIe+ vs. TBIe−: The number of SWDs per 24 h was 63 ± 87 in rats without epileptiform activity  and 316 ± 117 in rats with epilepsy .TBI+ vs. TBI−: The number of SWDs per 24 h was 76 ± 81 in rats without epilepsy (2 (range 5.61–76.38 mm2).Analysis of unfolded cortical maps drawn from histologic sections prepared 7 months post-TBI revealed the lesion core in the auditory association cortex  C. The me2, p < 0.01; Cohen’s d 1.02). No difference was detected between TBI rats with or without epilepsy  or epileptiform activity .Rats with PTZ-induced seizures at any timepoint had a smaller cortical lesion area than rats without PTZ-induced seizures , and survived the leave-one-out cross-validation with a misclassification rate of 36%   .Some of the individual parameters—particularly latency to the first ED and latency to the first PTZ-induced seizure—showed promise as biomarkers of epileptogenesis, differentiating the TBIe+ rats from the TBIe− rats .p = 0.05), and survived the leave-one-out cross-validation with a misclassification rate of 36%. On D180, the AUC was 0.752 (p < 0.05), with a misclassification rate of 32% , thus showing promise as diagnostic biomarkers of prior TBI , number of transitions to N3 (lights-on), total number of transitions (lights-on), and fragmentation index (lights-on)) resulted in an AUC of 0.792, and survived the leave-one-out validation with a misclassification rate of 28% as a diagnostic biomarker of prior TBI .p < 0.01) and an AUC of 0.847 during the lights-off period   .Although several combinations of sleep disturbance parameters showed promise as biomarkers for differentiating the TBIe+ and TBIe- groups, none of them survived the leave-one-out validation. Adding PTZ parameters did not improve the combinatory sleep biomarkers’ performance .Several individual sleep disturbance parameters showed potential as diagnostic biomarkers for differentiating rats with and without epilepsy . HoweverThe present study investigated whether seizure susceptibility increases over a period of weeks to months after TBI, and whether seizure susceptibility predicts the development of PTE in rats. Moreover, we examined whether TBI induces sleep disturbances—particularly in the N3 and REM periods, which are the most susceptible periods for seizure occurrence. Finally, we evaluated whether the PTZ test and/or the sleep parameters could provide biomarkers for prior TBI and ongoing epileptogenesis.Our previous studies in eight independent rat cohorts consistently demonstrated increased seizure susceptibility in the PTZ test at 3.5, 4, 6, 11, or 12 months after lateral FPI ,38,39,40In the present study, we administered PTZ four times. On the first testing day (D30), 30% of the rats in the sham group and 15% in the TBI group exhibited PTZ-induced seizures. We considered this acceptable for monitoring the later anticipated progression in seizure incidence. In fact, in the TBI group, the percentage of rats with PTZ-induced seizures was increased on the later testing days compared with that at D30. However, we found no progressively increasing differences between the sham and TBI groups in the average latency to the first spike, ED, or seizure, nor did the number of spikes, EDs, or PTZ-induced seizures, or the duration of the first seizure, differ between the groups on any testing day. This may relate to a large variability in seizure susceptibility between the rats at a given timepoint, as previously reported in , showingIn 18% of the vEEG-monitored TBI animals, unprovoked seizures were observed during the 7th post-TBI month—slightly less than the expected 25% ,51. UnliWe used the entire 24-h epoch sampled during the 7th post-injury month to analyze sleep disturbances in rats with TBI. The EEG epoch was carefully chosen from the recording period without preceding seizure occurrence. To our best knowledge, this is the longest epoch of EEG analyzed for sleep in rats, and the first study in animals with post-traumatic epileptiform activity.TBI induces chronic sleep disturbances in humans with TBIs, and in several animal models of TBI, including lateral FPI ,61,62,63As unprovoked seizures in rats with PTE almost exclusively occur during the N3–REM transition , we hypoOur study showed that a combination of three PTZ test parameters—including the latency to the first ED, number of EDs, and number of PTZ-induced seizures—formed a sensitive and specific diagnostic biomarker of the prior occurrence of TBI. Starting on D90, the combinatory biomarkers were also able to differentiate the rats with and without epileptiform activity, with an AUC of approximately 0.750, and with 77–79% accuracy. All of these parameters can be easily measured in EEG recordings, and are therefore expected to be reproducible in different laboratories.Several sleep parameters reporting on chronic sleep disturbances also demonstrated moderate sensitivity as biomarkers of the occurrence of prior TBI. The best biomarker performance was achieved by combining the average duration of REM sleep periods, number of transitions to N3, total number of transitions, and the fragmentation index, resulting in a moderate AUC of 0.792 and 77% accuracy.Like the individual PTZ test parameters, several individual sleep disturbance parameters  also differentiated rats with or without epileptiform activity or unprovoked seizures, with an AUC > 0.800. However, considering the small sample size, the data obtained should be considered preliminary. Moreover, the combinatory sleep disturbance biomarker approach did not reveal any biomarkers that survived the leave-one-out validation. In contrast to our expectations, biomarker performance was not improved by combining the PTZ test and sleep disturbance parameters.Overall, TBI animals show increased seizure susceptibility and sleep disturbance, which are further worsened in animals with epileptiform activity. The promising data on their potential as biomarkers for epileptogenesis warrant validation in a larger, independent animal cohort.The major difference between the present and previous studies in the lateral FPI model was that we assessed PTZ-induced excitability several times over the period of 6 months, whereas in previous studies the analysis was typically performed at only one timepoint see . AdditioRepeated administration of PTZ at a dose of 25 to 30 mg/kg can induce kindling in normal rats . Hamm et2, which is comparable to that in our studies with about 25% acute mortality [The acute post-impact mortality in the present study was 15%, which was less than the anticipated 25% typical for severe TBI ,69. Howeortality , suggestortality  reportedWe previously demonstrated in a large EPITARGET animal cohort that the average weight loss during the first week after the median 3.3 atm impact was 12% . In the In this study, we monitored the evolution of seizure susceptibility in a well-described rat model of PTE. Although rats with TBI showed indications and some evolution of reduced seizure susceptibility over the 6-month follow-up in the PTZ seizure-susceptibility test, the difference compared with the sham-operated experimental controls and progression over time were lower than anticipated. Nevertheless, some individual and combinatory PTZ test parameters showed promise as biomarkers of epileptogenesis, differentiating rats that developed epileptiform activity from those that did not. As we demonstrated previously, N3–REM transitions during sleep were associated with the occurrence of seizures and epileptiform activity. Consistent with this finding, sleep disturbance in rats with epileptiform activity or seizures after TBI manifested as a reduced duration of REM sleep periods and increased numbers of transitions, particularly from the N3 to awake stages, indicating sleep instability in this seizure-prone sleep period. Further analyses are warranted to assess the potential of sleep disturbance parameters as biomarkers of epileptogenesis in larger animal cohorts.


Mortality data and modifiable factors associated with urinary PGE-M were obtained from the parent cohort studies. Using linear regression models, we found that high urinary PGE-M levels were significantly associated with low education, heaving smoking, old age at urine collection, and abdominal obesity. Using Cox proportional hazards models, we found that increase (per standard deviation) of urinary PGE-M levels were significantly associated with overall mortality  and particularly deaths from cardiometabolic diseases . The increased death risks persisted across different time intervals during the follow-up and were stronger among participants who were younger than 60  at urine collection or perhaps among those who had higher education.Prostaglandins play a critical role in inflammatory response. To investigate the association of urinary PGE-M, a stable end-product of prostaglandin E2 (PGE PGE2 is one of the most abundant PGs produced in the body and is involved in all processes leading to the classic signs of inflammation. This metabolite is an important mediator of many biological functions, such as regulation of immune responses, blood pressure, gastrointestinal integrity, and fertility.Prostaglandins (PGs) play a critical role in the generation of the inflammatory response2 is an unstable compound that is rapidly metabolized in vivo to a stable metabolite, 11 a-hydroxy-9,15-dioxo-2,3,4,5-tetranorprostane-1,20-dioic acid (PGE-M). Thus, measurement of excreted urinary PGE-M has been used to quantify systemic PGE2 production in vivo. Urinary level of PGE-M is considered to have a more complete capture of prostaglandin production, as it will reflect a combination of prostaglandins from both the blood stream and the kidney3. We have previously reported the association between urinary PGE-M levels and multiple cancer risks.PGE6, including ischemic heart disease, stroke, and diabetes mellitus, which are the major causes of death in the world today7. Over the past decade, we have conducted multiple nested case-control studies13 to investigate the association between urinary PGE-M levels and multiple cancer risks and found the individuals with higher urinary PGE-M levels had increased risks of colorectal cancer8, gastric cancer12, breast cancer10, and pancreatic cancer11.In addition to cancers, it has become widely accepted that chronic inflammation is a driving force behind many other chronic diseasesIn addition, the detrimental effects of chronic inflammation on health are now known to persist throughout the life span to increase adulthood mortality. Characterizing the association of chronic inflammation with health risk and life span should thus also be a top public health priority. To this end, we investigated the modifiable factors associated with urinary PGE-M levels and test whether the latter were associated with premature death in this study.15. Briefly, participants were recruited from typical urban communities in Shanghai, China. The SWHS recruited 74,941 women aged 40–70 years from 1996 to 2000 with a 92.7% participation rate in the baseline survey. The SMHS recruited 61,480 men aged 40–74 years from 2002 to 2006 with a 74.0% participation rate in the baseline survey. At the time of enrollment, each participant signed consent and completed an in-person survey conducted by trained interviewers. Of the study participants, 65,754 (88%) women and 54,769 (89%) men provided a spot urine sample. Urine samples were collected into a sterilized cup containing 125 mg ascorbic acid to prevent oxidation of labile metabolites. After collection, the samples were kept in a portable Styrofoam box with ice packs (at approximately 0–4 °C) and processed within 6 h for long term storage at − 70 °C. All participants also filled out a biospecimen collection form at the time of sample collection, which included the date and time of sample collection, time of last meal, and use of any medications during the previous week.The 2927 non-cancerous adults included in this study consisted of controls from our previously case-control studies nested in the Shanghai Women’s Health Study (SWHS) and Shanghai Men’s Health Study (SMHS), two ongoing prospective cohort studies conducted in Shanghai, China. Mortality data and modifiable factors associated with urinary PGE-M were obtained from the parent cohort studies. The parent studies were approved by the institutional review boards of all participating institutions. We confirm that all methods were carried out in accordance with relevant guidelines and regulations. Detailed descriptions of study design and methods have been published elsewhereControls of our previous reported nested case-control studies were randomly selected from cohort members and individually matched to each case by age at sample collection (within 2 years), sex, time of sample collection (morning or afternoon), date of sample collection (within 1 month), and time interval since last meal (within 2 h). Controls were free of cancer at the time of cancer diagnosis of their corresponding case. The total sample from the previous nested case-control studies consisted of 5726 adults, including 2799 cancer cases  and 2927 non-cancer controls. We excluded all cancer cases from this study. Among the remaining 2927 non-cancer individuals, 2276 women and 651 men were participants from the SWHS and the SMHS respectively.13. Briefly, 0.75 mL urine was acidified to pH 3 with HCl and endogenous PGE-M was then converted to the O-methyloxime derivative by treatment with methyloxime HCl. The methoximated PGE-M was extracted, applied to a C-18 Sep-Pak, and eluted with ethyl acetate. Liquid chromatography was conducted on a Zorbax Eclipse XDB-C18 column attached to a ThermoFinnigan Surveyor MS Pump (Thermo Finnigan). For endogenous PGE-M, the predominant product ion m/z 336 representing [M-(OCH3 + H2O)]− and the analogous ion, m/z 339 [(M-OC[2H3] + H2O)]− for the deuterated internal standard, was monitored in the selected reaction monitoring (SRM) mode. Quantification of endogenous PGE-M used the ratio of the mass chromatogram peak areas of the m/z 336 and m/z 339 ions. The lower limit of detection of PGE-M was in the range of 40 pg, approximately 100-fold below levels in normal human urine. The coefficients of variation varied from 2.8 to 10.8% across the studies for samples analyzed within batches. Urinary creatinine levels were measured using a test kit from Sigma Company. The PGE-M levels were reported as ng PGE-M/mg creatinine. Laboratory staff was blinded to the case-control status of urine samples and the identity of quality control samples included in the studies.Urinary PGE-M  level was measured using a liquid chromatography/tandem mass spectrometric method as described in previous studiesDeath information was obtained through the follow-up surveys and by linkage to the database of the Shanghai Death Registry. The underlying of each death was assigned according to the international classification of diseases (9th version). The end date of the observation in this analysis was set as the date of death for deceased cohort members or the date of the last follow-up or June 30, 2016 for those who were still alive, half a year ahead of the last annual linkage with the Shanghai Death Registry, whichever was earlier. Cause-specific mortality categories were grouped according to the ICD-9 codes and were classified as diabetes (ICD-9 250), diseases of circulatory system (ICD-9 401-459), and all other causes.The distribution of urinary creatinine-adjusted PGE-M levels was skewed to the high values and thus log-transformation was used to improve the normality. We used linear regression models to evaluate changes of log-transformed PGE-M levels by selected demographic or lifestyle factors and medication use during the previous week of sample collection. Thus, the exponential of regression coefficients represents the ratio of geometric means for categorical variables as compared with the reference group or for each unit increment of a continuous variable. The selected covariates were included in the same linear models for mutual adjustment.16. The cause-specific mortality was analyzed in the competing risks context using the cause-specific hazards functions approach17. Potential confounders, including age at sample collection, education levels, income, use of NSAID or antibiotics one week before urinary sample collection, alcohol drinking status (ever/never), smoking pack-years, BMI and WHR, were included in the regression models for adjustment.The hazard ratios (HR) and 95% confidence intervals (CI) for the association of urinary PGE-M levels and subsequent overall or cause-specific mortalities were analyzed using Cox proportional hazards models stratified by batches of urinary PGE-M assay. We used the age as the time scale with left truncation at age of sample collection. The proportional hazards assumption was evaluated with the Schoenfeld residuals. We used the restricted cubic spline function for urinary PGE-M levels to examine the linearity of its association with the mortalitiesThe protocol was approved by the Committee of Ethical Research from the Vanderbilt University, and written informed consent was obtained from all participants.Among the 2927 participants included in this analysis, the median (range) age at urine sample collection was 59.7 (49.7–66.3) years and the median (range) of follow-up periods was 17.4 (14.3–18.6) years. A total of 361 (12.3%) participants died during the follow-up . Both diabetes and cardiovascular diseases are metabolic disorders and were thus were grouped together as cardiometabolic diseases in the subsequent analysis of cause-specific mortality.Table P = 0.9418 for all-cause mortality and P = 0.3737 for deaths from cardiometabolic diseases. Table P = 0.001 for all-cause mortality and HR = 1.27, 95%CI 1.11, 1.44, P = 0.0003 for death from cardiometabolic diseases). Urinary PGE-M levels were not significantly associated with deaths from the other causes.A significant linear association of urinary PGE-M levels with risk of all-cause mortality and deaths from cardiometabolic diseases was shown in Fig. P for heterogeneity test = 0.0014. A similar pattern was observed for the associations for deaths from cardiometabolic diseases with HR = 2.45, 95%CI 1.47, 4.07 vs. HR = 1.21, 95% CI 1.06, 1.38, P for heterogeneity test = 0.007. We also observed marginally significantly stronger associations  among participants who had higher education (> = high school) than among those who had less than high school education.As shown in Table The associations between urinary PGE-M levels and death were generally consistent across gender, NSAID use, smoking status, levels of waist-hip ratio, and different time intervals during the follow-up Table .Figure In this prospective study, we found that high concentrations of urinary PGE-M were positively related with low education, heaving smoking, old age at urine sample collection, and abdominal obesity as measured with waist-hip ratio. After adjustment for these covariates, we observed that higher concentrations of urinary PGE-M were still associated with a significantly increased all-cause mortality and particularly diabetes and CVD mortality. These positive association was consistent across different time intervals since study enrollment and were stronger among participants who were younger than 60 at urine collection or marginally significantly higher among those who had higher education.18, smoking20, older age22 and obesity24 were positively associated with chronic inflammation. The chemical constituents of tobacco smoke could induce cyclooxygenase-2 (COX-2) and PGE2 synthesis25. Aging and obesity are characterized by chronic inflammation. The inflammatory status becomes pervasive over time owing to the exposure to a variety of stressors in older people21. Obesity increases the release of pro-inflammatory cytokines and induces multiple components of an inflammatory state26. It is well established that biomarkers of inflammation are robust predictors of obesity and morbidity (chronic diseases)/mortality in older people28. Low socioeconomic status could affect inflammation due to chronic stress, obesity, unhealthy diet, and physical inactivity in people experiencing socioeconomic adversity18.Previous studies reported that low socioeconomic status1, PGE-M was observed to be positively associated mortality, mainly from cardiometabolic diseases in this study. It is well documented that inflammation plays an important role in the etiology of these diseases30. A recent study revealed that COX-2/PGE2 signaling was involved in the regulation of IL-1β auto-stimulation, thus forming an IL-1β/COX-2/PGE2 pathway loop, which may result in the high inflammation level and play a pivotal role in the pathogenesis of type 2 diabetes31. PGE2 plays a key role in the pathogenesis of cardiovascular diseases. It was reported29 that a deficiency of PGE2 synthase-1 reduced plaque burden in fat-diet low-density lipoprotein receptor knockout mice, and PGE2 synthase-1 knockout mice showed impaired left ventricular contraction after acute myocardial infarction. These findings indicate that PGE2 plays a pivotal role in cardiovascular inflammation.As an inflammatory marker8, gastric cancer12, breast cancer10, and pancreatic cancer11. Other studies also reported similar findings34. We did not include cancer patients in the current analysis, as their inclusion might introduce bias in the overall mortality analysis.We previously reported positive associations of PGE-M with multiple cancers including colorectal cancersWe observed consistent PGE-M-death association between early and late periods of the follow-up, which demonstrates the importance of chronic inflammation on premature mortality, emphasizing on controlling for chronic inflammation throughout the life span.35. Similarly, the increased health burden due to a constellation of biologic, behavioral, and psychosocial risk factors that are more prevalent in people with low socioeconomic status36. These factors hence should attenuate the influence of PGE-M on mortality.We found that the positive associations were weaker among participants who were older than 60 at urine collection or perhaps among those who had low education. Older people become more vulnerable a wide range of diseases due to declines in biological function and the accretion of cumulative risk factorsOur study’s strengths include its prospective design and adjustment for multiple potential confounders, which minimized the selection bias and confounding. In addition, our study has relatively large sample size and long period of follow-up time. To our knowledge, it comprises the first prospective study to directly evaluate the association of PGE-M with mortality with decent statistical power. Our study provided the direct evidence that PGE-M was associated with increased mortality. There may be a concern that the results from this study were based on a single measurement of a spot urine sample of PGE-M. Ideally, the study hypothesis would be bolstered if multiple measurements of urine samples collected at several different time points were available. Therefore, the association of PGE-M and mortality may have been underestimated due to the less than optimal exposure measurement.2.We found in this study that multiple modifiable factors such as low education, heaving smoking, and abdominal obesity were associated with increased inflammatory responses, as measured with urinary PGE-M concentrations. Higher concentration of urinary PGE-M was associated with increased mortality. We believe that improving lifestyles, e.g., stopping smoking and controlling body weight, would help decrease premature mortality through the pathway of decreasing over-production of PGE


Apostichopus japonicus achieves intestinal regeneration in a short period after evisceration, and multiple genes are involved in this process. The transcriptome of A. japonicus was screened for regeneration-associated protein (Aj-Orpin), a gene that is specifically upregulated during intestinal regeneration. The expression and function of Aj-Orpin were identified and investigated in this study. The 5′ and 3′ RACE polymerase chain reaction (PCR) was used to clone the full-length cDNA of Aj-Orpin. The open reading frame codes for a 164 amino-acid protein with an EF-hand_7 domain and overlapping signal peptides and transmembrane regions. Moreover, Aj-Orpin mRNA and protein expression during intestinal regeneration was investigated using real-time quantitative PCR and Western blot. The expression pattern of Aj-Orpin in the regenerating intestine was investigated using immunohistochemistry. The results showed that Aj-Orpin is an exocrine protein with two EF-hand-like calcium-binding domains. Expression levels were higher in the regenerating intestine than in the normal intestine, but protein expression changes lagged behind mRNA expression changes. Aj-Orpin was found to play a role in the formation of blastema and lumen. It was primarily expressed in the serosal layer and submucosa, suggesting that it might be involved in proliferation. These observations lay the foundation for understanding the role of Orpin-like in echinoderm intestinal regeneration. Apostichopus japonicus belongs to deuterostome, Echinodermata, a phylum at the branch between chordate and achordate, which indicates that it has special status in evolution. In response to environmental stress, it can eviscerate the intestine, respiratory tree, gonad, and all internal organs, followed by the recovery of functional internal organs in about three weeks [ee weeks . Due to ee weeks . The hisee weeks ,4. The bee weeks ,6,7. Aftee weeks ,10,11,12ee weeks ,13,14,15ee weeks ,13.Microvesicles and secretory vacuoles indicate that dedifferentiated cells have completed their redifferentiation and are forming intestinal luminal epithelium ,16. ThisTo investigate the mechanism of regeneration at higher throughput, researchers have constructed EST libraries ,19, geneA. japonicas [Holothuria glaberrima, the homologous gene Orpin showed no significant differential expression pattern in regenerating mesentery [According to transcriptome and expression profile studies, the regeneration-associated protein (Aj-Orpin) was involved in the intestinal regeneration of aponicas . The mRNesentery . AccordiA. japonicus and analyzed sequence characteristics to predict gene function in this study. Furthermore, we evaluated the levels of mRNA and protein expression, as well as the distribution of protein, at different stages of regeneration. Our findings lay the foundation for further research into the role of Orpin-like in echinoderm regeneration.However, the level of Aj-Orpin protein expression in intestinal regeneration has not been reported, nor has the level of mRNA expression in the early stages of regeneration. There have been no further studies on Orpin-like genes’ functions, so that Aj-Orpin could be the first regenerative-specific gene to characterize function. We cloned the full-length of Aj-Orpin gene from Aj-Orpin’s full-length cDNA sequence (GenBank accession number: ARI48335.1) was 1136 bp long and contained a 22 bp 5′-untranslated region (UTR), a 619 bp 3′-UTR, and a 495 bp ORF encoding a 164 amino-acid protein with a predicted MW of 17.68 kDa and a theoretical IP of 4.31. EF-hand_7 is a domain found in the protein sequence. According to García-Arrarás’s report [Saccoglossus kowalevskii (Hemichordata: Enteropneusta: Harrimaniidae). In A. japonicus, Holothuria glaberrima , and S. kowalevskii , Orpin genes were double-copied. A. japonicus PIK49419.1 shared 94.33% identity and 85% coverage with Aj-Orpin. Strongylocentrotus purpuratus  and Patiria miniata  both had three copies. Only one gene was discovered in Acanthaster planci . The above amino acid sequences were subjected to multiple sequence alignments and a CDD conserved domain search. The findings revealed that they all had an EF-hand_7 domain from the EF-hand domain pair, implying that they may have similar functions . Specific positions a were seA. japonicus. Sea cucumber , starfish , sea urchin (Strongylocentrotus purpuratus), and acorn worm  all had homologous sequences, indicating that the gene were found in echinoderms and hemichordates. A previous study discovered similar sequences in S. kowalevskii and sea cucumber H. glaberrima [S. purpuratus and the starfish A. planci and P. miniata were newly identified and added to the database. The homology percentage of all sequences was 28.28%, which was relatively low, mainly with high identity in the EF-hand conserved domain. No homologous genes have been found in vertebrates and other invertebrates. As confirmed in H. glaberrima [For the first time, this study cloned and identified the full-length cDNA of Aj-Orpin from the sea cucumber aberrima . The seaaberrima , Aj-Orpi2+) ion, forming a pentagonal bipyramidal array of seven oxygen ligands [2+ ion binding [When studying parvalbumin by X-ray diffraction, Kretsinger proposed the EF-hand model, which has a helix-loop-helix structure . The EF- ligands . EF-Hand ligands . Most kn binding ,39. Resi binding ,40. Oste binding ,42,43,44 binding , we hypo binding . In the A. japonicus at various regeneration stages were investigated. The transcription and translation levels of Aj-Orpin expression showed an inverse trend, possibly due to post-transcriptional regulation and the translation delay effect from mRNA to protein [Orpin A and Orpin B expression levels in the regenerative intestine and the normal intestine were similar in H. glaberrima, and the expression level in the mesentery decreased during the regeneration stage [H. glaberrima, mainly due to differences in sampling sites and the change in the proportion of mesentery and newborn intestine. However, the protein expression level increased gradually from 3 dpe to 14 dpe, peaking on day 14. This is consistent with immunohistochemical results, which show more expression at the serosal layer and submucosa at 7 dpe compared to 3 dpe according to the staining area. The expression pattern is similar with that of WntA, an important developmental gene as well as the patterns in cell proliferation [A. japonicus.The levels of Aj-Orpin mRNA and protein expression in the intestine of  protein . Orpin Aon stage . In our feration . Dediffeferation . The mesferation ,31,50, iA. japonicus adult sea cucumbers (70–100 g) were collected off the coast of Qingdao, Shandong Province. They were immediately transported to the laboratory and acclimated for two weeks at 15–17 °C before the experiment. Evisceration was induced by injecting 3–5 mL of 0.35 M KCl into the coelom [2 for ~1 h [e coelom . At eachfor ~1 h . Half ofTotal RNA was extracted from the intestine using the RNeasy Mini Kit and Oligotex mRNA Kit  according to the manufacturer’s instructions. The integrity and concentration of purified RNA were determined using 1% agarose gel electrophoresis and Nanodrop1000 .TM RACE cDNA Amplification Kit , and 5′ and 3′ rapid amplification of cDNA ends polymerase chain reactions (RACE PCR). The manufacturer’s protocol was followed to obtain 5′- and 3′- RACE-Ready cDNA. Primer3  was used to design gene-specific primers (GSP)  based on the sequence fragments obtained from the transcriptome library [Aj-Orpin full-length cDNA was obtained from the SMARTerUsing 1% agarose gel electrophoresis, the 5′ and 3′ PCR products were detected, and gel extraction was performed . Following the protocol, the PCR products were inserted into the pMD19-T vector (Takara) and transformed into JM109 competent cells (Takara). The transformed competent cells were resuscitated and cultured overnight on Luria–Bertani solid medium containing 100 μg/mL ampicillin. Positive clones were selected and cultured in a liquid SOC medium for 12–16 h and then sequenced by HUADA . To obtain the full-length cDNA of Aj-Orpin, DNAStar software  spliced the 5′ end sequence, known fragments, and 3′ end sequence.https://www.ncbi.nlm.nih.gov/orffinder/, accessed on: 1 March 2022) was used to predict amino acid sequence and find an open reading frame (ORF) based on the full-length cDNA sequence. The Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) was used to analyze sequence homology . The amino acid sequence, as well as the molecular weight (MW), theoretical isoelectric point (PI), and transmembrane region, were analyzed using the ExPASy server . SignalP  was used to predict signal peptides. The conserved protein domain family was predicted using NCBI’s CDD [ORFfinder  was used on an Eppendorf MastercyclerHep realplex  to assess Aj-Orpin mRNA expression levels at different regeneration stages. The reaction system was set up according to the instructions, with predenaturation at 95 °C for 5 s, followed by 40 cycles of denaturation at 95 °C for 10 s, 60 °C renaturation for the 20 s, and extension at 72 °C for 30 s. The melting curve was added as the final step to ensure that the primers were specific. The expression level of Aj-Orpin mRNA was measured using the 2−ΔΔCt method.Total RNA extraction and quality verification from the intestine at various stages of regeneration were performed using the methods mentioned above. Using Prime Script™ RT Reagent Kit with genomic DNA Eraser (Takara), RNA was reverse transcribed into single-stranded cDNA, which was used as the template for qRT-PCR. We used NADH dehydrogenase as a reference gene . Table 1Total protein content was extracted from 15 mg intestine tissue, mixed with an equal volume of 2× loading buffer, and then denatured in boiling water for 5 min. We slowly added 15 μL of the mixture into the sample tank to carry out polyacrylamide gel electrophoresis until the bromophenol blue loading buffer migrated to the bottom of the gel (the 15% of the resolution gel and the 5% of the stacking gel), then transferred to PVDF membranes. Phosphate-buffered saline/0.1%Tween-20 (PBST) was used to wash the membrane three times. To block for 2 h at room temperature, 1% casein was used, then incubated with rabbit anti-Aj-Orpin antibody diluted 1:1000 with 1% casein and commercial β-actin antibody diluted 1:5000 with PBST. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G was used as the secondary antibody . The film was exposed, developed, and fixed in a dark room. The protein expression level was normalized by comparing the density of immunoreactive bands using β-actin. Image J was used to perform the quantification analysis. The primary polyclonal antibody anti-Aj-Orpin was obtained by immunizing rabbits with synthetic specific polypeptide (CGDEKISWQEFPDMN). β-actin antibody, as a loading control, was provided by GenScript  and the procedure was completed at the GenScript biological company  for Western blotting and following immunohistochemistry.To remove endogenous catalase, intestinal tissue sections at various stages of regeneration were dewaxed and rehydrated, then treated with 3% hydrogen peroxide for 10 min. The sections were antigen-repaired in sodium citrate buffer using a pressure cooker. They were washed in phosphate-buffered saline (PBS) and then blocked with 10% bovine serum albumin at 37 °C for 30 min after cooling to room temperature. After washing with PBS, the sections were incubated with a primary antibody against Aj-Orpin (1:100) at 4 °C overnight. The control group used PBS instead of primary antibody. The samples were then incubated with a peroxidase-conjugated secondary antibody (1:500) for 30 min at 37 °C. The color reaction was carried out using a diaminobenzidine substrate kit . Finally, the samples were redyed with hematoxylin, dehydrated with gradient alcohol, sealed with neutral balsam, and photographed under a microscope.n = 3) with a difference significance level of p < 0.05.Using the statistical package for social sciences, a one-way analysis of variance was used for statistical analysis. GraphPad Prism 9.0.0.121 was used to create histograms. All data are presented as the mean ± standard error  and lumen formation (7–14 dpe). Aj-Orpin was highly expressed in the serous layer and submucosa, suggesting that it may work in tandem with cell proliferation. Additional research is needed to fully understand Aj-Orpin’s role in intestinal regeneration.In this study, we cloned and characterized the full-length cDNA of Aj-Orpin, which was 1136 bp long and contained a 22 bp 5′-UTR, a 619 bp 3′-UTR, and a 495 bp ORF encoding a 164 amino acid protein. Aj-Orpin was discovered to be a two-copy gene found only in echinoderms and


Mycobacteria tuberculosis (Mtb) infection is important for the pathogenesis and final clearance of intracellular Mtb infection. In addition, it is valuable for the diagnosis of tuberculosis. In this pioneering work, we tested in vitro and in vivo antigen presentation and diagnostic application of a recombinant overlapping peptide-protein derived from two Mtb RD1 antigens ESAT-6 and CFP-10 (ROP-TB). The overlapping peptide sequence of ROP-TB is cleaved by the cathepsin S enzyme and covers the entire length of the two proteins. ROP-TB can be expressed and purified from E. coli. Once taken in by antigen-presenting cells, ROP-TB can be cleaved into a peptide pool by cathepsin S within the cells. We found that in dendritic cells, ROP-TB can be processed in 6 hours of co-culture, while the ESAT-6/CFP-10 fusion protein remained in the endosomal compartment. In Mtb-infected mice, ROP-TB stimulated stronger specific T cell responses than pooled synthetic peptides derived from ESAT-6 and CFP-10. With regard to the presentation of in vivo antigens, in a guinea pig model infected with Mtb, ROP-TB induced delayed type hypersensitivity (DTH) responses comparable to those of the tuberculin purified protein derivative (PPD) and ESAT-6/CFP-10 fusion protein. In Mycobacterium bovis (Bovine TB)-infected cattle, ROP-TB elicited DTH responses. Finally, in Mtb infected patients, ROP-TB stimulated cellular immune responses in majority of patients (16/18) of different HLA phenotypes while a single peptide derived from the same proteins did not elicit the immune responses in all patients. In summary, in vitro and in vivo data suggest that ROP-TB stimulates a strong cellular immune response irrespective of HLA phenotypes and is therefore suitable for use in vitro and in vivo diagnostics.Cellular immunity in  Cell-mediated immunity requires antigen presentation by class I or II MHC molecules on the surface of antigen-presenting cells (APC). CD8+ T cells typically recognize epitopes presented with MHC class I derived from endogenous antigen degradation in the cytoplasm of any nucleic cell, including APCs (Class I pathway). In contrast, exogenous antigens are processed in the endosome/lysosome in APC into peptides which can be presented by MHC class II (only expressed in APCs) to the cell surface and stimulate CD4+ T cells (Class II pathway). In some situations, exogenous antigens can be cross-presented in APCs to prime CD8+ T cells (Cross-presentation). However, the efficiency of antigen cross-presentation is low –3.Mtb infection. Furthermore, since the antibody is not suitable for the diagnosis  has recommended two methods based on cellular immune response for latent TB infection (LTBI)  release assay (IGRA) has also been suggested as an accessory diagnostic method for active TB  . The metctive TB .Mtb antigens. If the individual has LTBI, memory T cells in the PBMCs will be activated in vitro by Mtb antigens to release cytokines, including IFN-γ, which can be measured (Mtb RD1 antigens: early secreted antigen target 6 (ESAT-6) and cell filtrate protein 10 (CFP-10). The advantages of using the ESAT-6 and CFP-10 derived peptide antigens are: 1) distinguish from BCG  vaccination; 2) stimulate strong cellular immune responses; and 3) overcome MHC restriction as the pooled peptides contain multiple epitopes suitable for different HLA phenotypes. However, quality control in the industrial manufacturing process of multiple peptides requires multiple GMP processes and is labor-intensive and time-consuming. It has posed a limitation to cost-effective manufacturing of diagnostic kits. Consequently, the use of the IGRA assay as a screening test for latent TB for the general population has not become a reality.The first cellular immune response-based method is the IGRA, in which peripheral blood mononuclear cells (PBMCs) of an LTBI-suspected individual are exposed to measured . The keyin vivo method based a delayed-type hypersensitivity (DTH) response in which the antigen presentation of the exogenous PPD antigen leads to a cellular immune response as a consequence of a collective cytokine release. The sign of an immune response is a skin reaction at the PPD injection site. The advantages of this method include the ability to measure cellular immune responses not only based on one cytokine  but also on the collective stimulation of other factors (cytokines/chemokines). Furthermore, it a simple and economical test, with no need for in vitro cell culture equipment or expensive reagents (tissue culture plates and antibodies); therefore, it is suitable as a screening test in the general population. However, the PPD-based skin test cannot distinguish Mtb infection from BCG vaccination.Another method of detecting LTBI is the Mantoux skin test based on a purified protein derivative (PPD). This is an Mtb infection from BCG vaccination and be suitable for industrial manufacture. We have previously reported using recombinant overlapping peptide (ROP) proteins as stimulants for cellular immunity  was interspersed between each peptide. Cathepsin S is known to exist in professional APCs (10). A schematic presentation of ROP-TB is shown in To remove any potential endotoxins that may have potentially contaminated the product, ROP-TB was further purified by endotoxin removal chromatography. Subsequent endotoxin levels in ROP-TB before application in cell cultures was tested and were <1 EU/mL. To test the effects of endotoxin on the ROP-TB-based IGRA assay, an enzyme-linked immunosorbent spot (ELISPOT) assay study using ROP-TB spiked with endotoxin was carried out. The doses of endotoxin used (up to 33 EU/mL) did not influence the results of the ROP-TB-related ELISPOT .in vitro with cathepsin S. After digestion, samples were analyzed using SDS-PAGE. As in vitro into peptides of different sizes by cathepsin S  by tracing their intracellular localization at different time points in the mouse dendritic cell line DC2.4 cells. ROP-TB, TB peptides, and TB protein were labeled with a green fluorescent dye (WV=530/30nm) and were incubated with DC2.4 cells for 30 min and 6 h. DC2.4 cells were co-incubated with Lysotracker to emit red fluorescence (WV=630/30nm). The uptake of ROP-TB, TB peptides, or TB protein is shown in green, and endolysosomes are shown in red under a confocal microscope. The co-localization between red and green colors represents the approximate location of the two objects. As shown in Mtb peptides ex vivo in a mouse model. We first established the test model by infecting BALB/c mice with Mtb (H37R1) intranasally . As shown in Spleen cells from in vivo. To investigate whether ROP-TB can be processed and presented in vivo to specific T cells, guinea pigs were infected with Mtb H37R1, and DTH-related skin reactions to ROP-TB, ESAT-6/CFP-10, and PPD (purified protein derivative) were investigated. Both ROP-TB and ESAT-6/CFP-10 trigger positive responses to DTH, as well as PPD (Delayed-type hypersensitivity (DTH) reflects T cell responses to antigen stimulation M. Bovis) epidemics have been reported in many parts of the world. This has posed a risk for humans drinking infected, unpasteurized milk. Practically all cattle on a farm need to be slaughtered even if only one head of cattle is found to be infected with M. bovis. This causes great economic loss. Thus, as a preventative measure, a tuberculin-based skin test using a PPD is usually performed to screen for infected cattle  recruited for ROP-TB and PPD- based skin tests. As shown in Since equences , we wondTo observe whether ROP-TB can promiscuously stimulate the memory T cell response in humans, we tested ROP-TB for a specific T cell immune response measured by ELISPOT in a population of diverse HLA phenotypes. We recruited 18 patients with tuberculosis who were diagnosed by sputum test, acid-fast staining, and lung computed tomography (CT) scan. The demographic data for these patients are summarized in Monitoring and detecting specific T cell responses is important for many diseases, such as tumors, infectious diseases, and inflammatory diseases. However, currently, TB is the only disease indication for commercially available, T cell-based diagnostic assays, which consist of a skin test and IGRA assays. The difficulties of developing a specific T-cell-based test include the identification of specific antigens that can promiscuously stimulate T cells from various MHC backgrounds; the manufacture of such antigens without labor intensive processes such as multiple GMP manufacturers and/or quality controls; and the requirement of cell culture facilities for their execution, which may hamper the use of the assays in a hospital setting, especially in remote areas in developing countries.For the two TB diagnostic methods currently used clinically, the key reagents/antigens are derived from different origins. The antigen used in the skin test assay is a PPD, while the antigen used in the IGRA assays consists of a peptide pool derived from ESAT-6 and CFP-10 , 17. PPDE. coli system (The aim of this study was to develop an antigen suitable for both IGRA and a diagnostic skin test. We designed a chain of overlapping peptides (ROP) linked by the enzyme substrate cathepsin S. This antigen is cleavable in APCs where the enzyme cathepsin S is located. After degradation by the enzyme, the antigen will undergo presentation and/or cross-presentation to stimulate T cells. Our data from this study showed that ROP-TB could be produced and purified recombinantly using the i system Figure 1E. coli, which can be adapted to large-scale manufacturing processes, the costs are reasonably low. Moreover, ROP-TB is produced as one entity; therefore, only one quality control step is required in the downstream process of manufacture. Furthermore, unlike pooled peptides, ROP-TB is convenient (as it is applied as a single substrate), which is useful when applying for regulatory approval. Third, like pooled peptides, the antigen is promiscuous and stimulates T cells from individuals of different HLA phenotypes. This has been demonstrated by this study . A single colony was harvested in LB medium (50 μg/mL Kanamycin) and cultured overnight. Bacteria from the overnight culture were then inoculated into the new LB medium at a 1:100 ratio and cultured until the OD600 reached 0.6. Isopropyl β- d-1-thiogalactopyranoside  was added at a final concentration of 0.5 mM and the bacteria were collected by centrifugation after 16 h of incubation. Lysis buffer  was used to resuspend the bacteria, which were then lysed by sonication. The insoluble fractions were separated by centrifugation at 15,000 g for 1 hour.For purification, the corresponding plasmids were transformed into The protein was further purified by nickel affinity chromatography using Ni-NTA . To detect expressed soluble TB protein (ESAT-6-CFP-10), Ni-NTA resin was added to the soluble fractions for 1 h. The protein was washed with lysis buffer containing 10 mM imidazole (washing buffer) and eluted with lysis buffer containing 300 mM imidazole (elution buffer). The insoluble ROP-TB in the pellet was resuspended in 8 M urea buffer and centrifuged at 15,000 g for 1 h. The rest procedures were similar to those of ESAT-6-CFP-10 except that the washing and elution buffer contained 8 M urea. For refolding, the eluted proteins (ESAT-6-CFP-10 and ROP-TB) were exchanged by PBS for dialysis.Ni-NTA purified ROP-TB was further purified by endotoxin removal chromatography . Briefly, the column (1.5 mL) was washed with balanced buffer, then ROP-TB was flowed through the column at 0.5 mL/min and the effluent collected.The endotoxin present in ROP-TB was detected using the Limulus amebocyte lysate assay  following the manufacturer’s instructions. Briefly, ROP-TB was diluted with endotoxin-free water by serial two-fold dilutions, then diluted ROP-TB was added, respectively, to the Limulus amebocyte lysates, which were dissolved in 100 μL endotoxin-free water. The mixtures were incubated in ampoule bottles at 37°C for 1 hour. If a gelatinous substance formed at a certain dilution, it was considered positive: Endotoxin concentration was calculated as maximum positive × 0.125 EU/mL, which indicated the sensitivity of Limulus amebocyte lysate.Purified protein (ROP-TB) in PBS and 4mM DTT was incubated with Cathepsin S  at 37°C for 0.5, 1, 2, 4, 8, and 22 h. The mass ratios of enzyme and protein in digestion were 1:50 and 1:100, respectively. Samples with different digestion times were run through SDS-PAGE for measuring enzyme activity.5 per dish) at 37°C for 30 minutes or 6 hours. ROP-TB, TB peptides, and TB protein marked with the LIVE/DEAD cell stain kit . To stain endolysosomes, 10 μL of 1:200 diluted (red) Lysotracker  was added to the cell culture one hour before incubation. After incubation, cells were washed and fixed with 4% paraformaldehyde for 10 min. The samples were further stained with DAPI  prior to observation under a confocal microscope .To observe antigen uptake, 1 μM ROP-TB, TB peptides, and TB protein (ESAT-6-CFP-10) were incubated with DC2.4 cells  in culture plates (3x10Mtb (H37R1) at 5x105 CFU in 40 μL. After infection, the animals were housed in cages kept in laminar flow safety enclosures in a Level III biosafety facility.The mouse experiment was outsourced to the Shanghai Public Health Center . All procedures were approved by the Committee of the Shanghai Public Health Center and followed the national guidelines for the use of experimental animals. Briefly, all mice  were infected intranasally with Mtb model, the lung tissues of infected and uninfected mice were sectioned for fast acid staining. The sections were placed in a staining dish and stained with Carbol Fuschin. After heating to steam and incubation for 5 min, the lung sections were washed with slow running water, decolorized with acid alcohol, counterstained with Malachite green for 1 min, and then air-dried. The final sections were positive when red and negative otherwise. The procedure of H&E staining (Sigma)for the evaluation of lymphocyte infiltration is the following: samples were first fixed overnight with 4% paraformaldehyde. After embedding in paraffin, the samples were cut into 2-μm thick sections and subjected to H&E staining. Histopathological analysis was performed using a Zeiss microscope.To verify the establishment of the 5 cells/well with 5 μg/well ROP-TB or peptides at 37°C for 24 h. Then, 96-well culture plates were precoated with IFN-γ antibodies. After the incubation, the cells were discarded, and the plates were washed with PBS followed by incubation with biotinylated anti-IFN-γ antibodies for 2 h at room temperature. The plates were washed with PBS and an enzyme-labeled anti-biotin antibody was added for 1 h. Finally, BCIP/NBT was used for color development, and the reaction was stopped by washing plates with tap water. Spots were counted with an Elispot reader .The IGRA-based T cell function assay was performed using ELISpot kits  . BrieflyThe human experiments were reviewed and approved by the Ethics Committee of Changzhou No. 3 People’s Hospital. Eighteen patients with tuberculosis infection were recruited from the hospital and provided informed written consent prior to the collection of their detailed clinical data Table 2.5/150 μL of PBMCs were stimulated overnight with 5 μg/well of ROP-TB, either pooled or as singular peptides in anti-IFN-γ-Ab precoated plates . Cells were discarded and biotinylated anti-IFN-γ antibodies were added for a 2-h incubation at room temperature followed by another 1-h incubation at room temperature with an enzyme-labeled anti-biotin antibody. After the color development, the reaction was stopped by washing the plates with tap water, and the plates were air dried. Spots were counted with an Elispot reader .IGRA-based T cell testing was performed using ELISpot kits . Briefly, 2.5x10Mtb (H37R1) of 0.5 mL (5x104 CFU) through nasal drop. After infection, the animals were housed in cages contained within laminar flow safety enclosures in a Level III biosecurity facility.The experiment was outsourced to the Shanghai Public Health Center . All animals were infected with The DTH-based skin test was performed as follows: guinea pigs were divided into six groups of five animals per group. They were injected intradermally on the flank with 0.1 mL of ESAT-6-CFP-10 , ROP-TB , national standard and negative control . The diameters of the erythema were read after 24–72 hours.M. bovis TB, to be compared with ROP-TB in the experiments. The PPD skin test was performed following the standard Chinese diagnostic technique for tuberculosis in animals. ROP-TB antigen  was injected into a volume of 0.1 mL on one side of the cow neck, while PPD  on the other side. Skin thicknesses at injection sites were measured between 48 and 72 h of injection. For the readout of the skin test, if the skin thickness of the cattle was ≥ 4 mm, the animal was considered skin test-positive and negative if the thickness was <2 mm. In the case that the difference was nearly or equal to 2 mm, the result was considered inconclusive.The experiments were approved by the Ethics Committee of Southwest University . Cattle from two farms  were recruited. To evaluate the potential of a DTH-based skin test with ROP-TB, we selected commercially available PPD, which is the standard reagent for the diagnosis of Statistical analysis was performed with Student’s t test. P values <0.05 were considered statistically significant. The original contributions presented in the study are included in the article/The human experiments were reviewed and approved by the Ethics Committee of Changzhou No 3 People’s Hospital. The patients/participants provided their written informed consent to participate in this study. The mouse and guinea pig experiments were approved by the Committee of Shanghai Public Health Centre and the cattle experiments were approved by Ethic Committee of Southwest University. Written informed consent was obtained from the owners for the participation of their animals in this study.SJ, WL, and JW designed the study and wrote the manuscript; QZ performed most of the experiments and carried out data analysis; XL performed the cow skin test and ELISPOT assays; ST helped to carry out the HLA analysis; DC performed the purification of ROP-TB; YG and LG helped to collect patient samples; RZ participated in the experimental design and management. All authors contributed to the article and approved the submitted version.The authors declare that this study received partial funding from Oxford Vacmedix UK Ltd and CBI. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication. This research was also supported by National Key Research and Development Program of China (2018YFE0192500) and the Key Research and Development Program of Changzhou . SJ is funded by Innovate UK.QZ, XL, YG, DC, RZ, WL and SJ are employees and/or shareholders of Oxford Vacmedix (Changzhou) Ltd which develops TB diagnostic kits. WL is employed by Shanghai JW Inflinhix Ltd (a subsidy company of Oxford Vacmedix(Changzhou)).The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.